.beta.-Interferon has been well known as a tumor cytotoxic factor which is produced by human derived fibroblasts. Physiologically active factors produced by fibroblast cells have been disclosed in Japanese Laid-open Patent Application Nos. 146,293 (1983), 1,872 and 33,120 (1986), 103021 (1987) and 10998 (1989). On the process of investigation of antitumor protein derived from fibroblasts, the inventors of the present invention have found antitumor protein, being quite different from proteins previously reported and succeeded in cloning the cDNA coded for the novel protein. The inventors identified total amino acid sequence and confirmed the usefulness thereof. The novel antitumor protein and gene thereof were disclosed in WO 90/10651 and named TCF-II. Total amino acid sequence of the protein deduced from the cDNA sequence is shown in Sequence Table No. 1.
TCF-II has both potent antitumor activity and growth stimulation activity for normal cells, and was confirmed as a member of the family of hepatocyte growth factor (HGF). TCF-II shows molecular weight of 78,000.+-.2,000 or 74,000.+-.2,000 in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Under reducing conditions, TCF-II shows a common A band with molecular weight of 52,000.+-.2,000 and a band B with molecular weight of 30,000.+-.2,000 or a band C with molecular weight of 26,000.+-.2,000. The monoclonal antibodies against TCF-II had been disclosed in Japanese Laid-open Patent Application No. 97 (1993) and the inventors of the present invention found that the antibodies equally recognize both TCF-II and HGF during the investigation of the present invention.
TCF-II and HGF have different biological activities and their differentiation is necessary. However, TCF-II and HGF have very similar amino acid sequences and their respective antibodies exhibit cross reactivity each other. No specific epitope of TCF-II has been obtained, therefore, no monoclonal antibody which exhibits affinity solely with TCF-II without any affinity with HGF has been obtained.
The present inventors noticed the usefulness of TCF-II and have been investigated the application to an antitumor agent and a wound healing agent, and the use as a marker for the diagnosis of diseases.
There is a marked difference in biological activities between TCF-II and HGF. HGF is respectively about 28-, 10- and 2-fold more potent than TCF-II in specific activity for stimulating growth of human umbilical vein endothelial cells (HUVEC), human aorta smooth muscle cells (AOSMC), and murine myeloblastic cell line (NFS-60) (Goto et al.: SEIKAGAKU published by the Japanese Biochemical Society, 65(8) 835, 1993). While, TCF-II is respectively about 1.4- to 1.9-fold more potent than HGF in specific activity for stimulating growth of rat hepatocytes (Shima et al.: Biochem. Biophys. Res. Commun., 180, 1,151-1,158, 1991; Matsumoto et al.: ibid., 181, 691-699, 1991) and about 3- fold more potent than HGF in specific activity for stimulating growth of pig kidney epithelial cells (LLCPK-1) (Goto et al.: SEIKAGAKU, 65(8) 835, 1993, published by the Japanese Biochemical Society). Therefore, selective and quantitative determination of TCF-II and HGF is very useful to investigate their excretion ratio in human cell culture, and differences in their secretion sites and physiological roles in body, and the relationship between the blood concentrations of TCF-II or HGF, and disease conditions.
However, TCF-II and HGF have high homology each other, and selective and quantitative determination of TCF-II in samples containing both TCF-II and HGF such as human cell culture, serum or plasma can be carried out only by a bioassay using difference of biological activity in target cells between TCF-II and HGF as described above. Furthermore, the accurate and precise quantitative determination was difficult by the bioassay. At present, immunoassay is a main method for the determination of very small amount of substances and an antibody suitable for the selective determination of TCF-II, that is, a monoclonal antibody which reacts only with TCF-II, but not with HGF, has been desired. The detection of secretion sites of TCF-II in human tissue can be effectively performed by application of such an antibody, and affinity chromatography using such antibody makes it possible to specifically purify and effectively recover TCF-II from human cell culture containing both TCF-II and HGF.
However, TCF- N and HGF have similar amino acid sequences. Heretofore, conventional immunization of an animal to provide antibody producing cells, followed by cell fusion to prepare hybridoma could not screen a cell line for the production of an antibody which selectively recognize TCF-II. Thus, no antibody which recognize only TCF-II has been obtained.